Radiotherapy is an effective treatment for most tumor types. However, emerging evidence indicates an increased risk for atherosclerosis after ionizing radiation exposure, initiated by endothelial cell dysfunction. Interestingly, endothelial cells express connexin (Cx) proteins that are reported to exert proatherogenic as well as atheroprotective effects. Furthermore, Cxs form channels, gap junctions and hemichannels, that are involved in bystander signaling that leads to indirect radiation effects in non-exposed cells. We here aimed to investigate the consequences of endothelial cell irradiation on Cx expression and channel function. Telomerase immortalized human Coronary Artery/Microvascular Endothelial cells were exposed to single and fractionated X-rays. Several biological endpoints were investigated at different time points after exposure: Cx gene and protein expression, gap junctional dye coupling and hemichannel function. We demonstrate that single and fractionated irradiation induce upregulation of proatherogenic Cx43 and downregulation of atheroprotective Cx40 gene and protein levels in a dose-dependent manner. Single and fractionated irradiation furthermore increased gap junctional communication and induced hemichannel opening. Our findings indicate alterations in Cx expression that are typically observed in endothelial cells covering atherosclerotic plaques. The observed radiation-induced increase in Cx channel function may promote bystander signaling thereby exacerbating endothelial cell damage and atherogenesis.